循环微囊泡miR-27a参与脑缺血小鼠血脑屏障紧密连接损伤的机制研究

Effects of circulating microvesicles containing microRNA-27a on blood brain barrier tight junction injury of ischemia stroke mice

目的 研究脑缺血小鼠循环系统中,循环微囊泡miR-27a对血脑屏障紧密连接损伤的作用机制。方法 构建小鼠大脑中动脉栓塞模型,离心超滤法分离缺血2 h脑组织上清液中微囊泡,采用透射电镜观察微囊泡形态,并检测微囊泡直径。在脑缺血2 h模型基础上,股静脉注射5 mg·kg^(-1)微囊泡,TTC染色观察缺血脑组织梗死体积;HE染色检测紧密连接蛋白occludin和claudin-5变化;Western blot检测occludin、claudin-5、TLR4、NF-κB、p38蛋白表达水平;ELISA法测定IL-1β和TNF-α含量。结果 从缺血小鼠脑组织中分离的微囊泡为圆形或近圆形的双层膜结构小囊泡,颗粒平均直径为160 nm,符合微囊泡的形态学特征。与缺血组比较,注射微囊泡miR-27a能够加重缺血小鼠脑组织损伤,进一步增加脑组织梗死体积,降低缺血脑组织中occludin和claudin-5蛋白表达,上调TLR4表达,激活NF-κB、p38蛋白的磷酸化,IL-1β和TNF-α释放增多;而给予阻断剂antagomiR-27a能够减轻小鼠脑组织损伤,抑制TLR4、NF-κB、p38蛋白的活化。结论 微囊泡miR-27a可明显增加缺血小鼠脑组织损伤,加重缺血脑组织紧密连接损伤,其机制与上调缺血脑组织中TLR4、NF-κB、p38蛋白的磷酸化,释放IL-1β和TNF-α相关。

Aim To study the effect of circulating microvesicles containing miR-27 a on blood brain barrier tight junction injury of ischemia stroke mice and its mechanism. Methods The middle cerebral artery occlusion mouse model was established,and the microvesicles in the supernatant of 2 h of ischemic stroke brain tissues were separated by centrifugal ultrafiltration method. The transmission electron microscopy was used to observe microvesicle morphology,and the diameter of microvesicles was detected. Based on the 2 h ischemia stroke mouse model,mice were injected via femoral vein with microvesicles at 5 mg · kg^（-1）. TTC staining was applied to detect infarction volume of ischemia brain,while HE staining was applied to detect the expression change of tight junction protein occludin and claudin-5. Western blot was subjected to detect occludin,claudin-5,TLR4,NF-κB and p38 protein expression. ELISA method was used to measure the contents of cytokines IL-1β and TNF-α. Results The microvesicle shape was approximately circular bilateral membrane structure,with an average diameter of 160 nm,which conformed to the morphological characteristics of microvesicles. Compared with the ischemic stroke group,injection of microvesicles could aggravate the damage of brain tissues in ischemic mice,further increase the infarct volume and reduce the positive staining areas of occludin and claudin-5 in ischemia brain tissues. Meanwhile,the protein expressions of occludin and claudin-5 further decreased,and further up-regulated TLR4 and phosphorylation of NF-κB and p38 compared with the ischemia stroke group. Also,more content of IL-1β and TNF-α were detected in ischemia stroke group injected with microvesicles compared with those in ischemia stroke group with significant difference,while the injection of antagomir-27 a could alleviate brain damage and reduce the activation of TLR4,NF-κB and p38 in ischemia stroke mice.Conclusions Microvesicles containing miR-27 a could significantly attenuate brain injury in ischemia stroke mice,while aggravate the tight junction damage of ischemia brain. The mechanism might be correlated with the up-regulation of the expression of TLR4,the phosphorylation of NF-κB,p38,and the release of cytokines IL-1β and TNF-α.